The human genome project has succeeded in sequencing most regions of human DNA. Work to identify the genes and sequence alterations associated with disease continues at a rapid pace. Linkage studies are used to associate phenotype with genetic markers such as simple sequence repeats or single nucleotide polymorphisms (SNPs) to identify candidate genes. Sequence alterations including SNPs, insertions, and deletions that cause missense, frameshift, or splicing mutations then may be used to pinpoint the gene and the spectrum of responsible mutations.
However, even when the genetic details become known, it is often difficult to use this knowledge in routine medical practice, in large part because the methods to analyze DNA are expensive and complex. When costs are significantly lowered and the methods dramatically simplified, it is expected that DNA analysis will become accessible for use in everyday clinical practice for effective disease detection and better treatment. Ideal DNA analysis is rapid, simple, and inexpensive.
When a disease is caused by a limited number of mutations, or when a few sequence alterations constitute a large proportion of the disease cases, direct genotyping is feasible. Traditional methods range from classical restriction digestion of PCR products to closed-tube fluorescent methods. Closed-tube methods of DNA analysis can be simple to perform. Once PCR is initiated, no further reagent additions or separations are necessary. However, when one allele is present in small quantities, that allele may be difficult to detect.
Examples of methods that may be used to amplify target oligonucleotide sequences, such as DNA sequences, include the TaqMan assay, a homogenous assay for detecting polynucleotides (see U.S. Pat. No. 5,723,591). In this assay, two PCR primers flank a central probe oligonucleotide. The probe oligonucleotide contains a fluorophore and quencher. During the polymerization step of the PCR process, the 5′ nuclease activity of the polymerase cleaves the probe oligonucleotide, physically separating the quencher, which increases fluorescence emission. As more PCR product is created, the intensity of fluorescence emission increases.
Molecular beacons are another method for the detection of polynucleotides (see U.S. Pat. Nos. 6,277,607; 6,150,097; and 6,037,130). Molecular beacons are oligonucleotide hairpins with a fluorophore/quencher pair, and the oliogonucleotide undergoes a conformational change when it binds to a perfectly matched template. The conformational change increases the distance between the fluorophore and the quencher, which increases the fluorescence emission from the fluorophore.
Another known method for target sequence detection uses a pair of probes, one of which has an acceptor fluorophore and the other a donor fluorophore, and the probes hybridize to adjacent regions of the target sequence. After amplification of the target sequence, the two probes hybridize with the target sequence and the donor fluorophore interacts with the acceptor fluorophore to generate a detectable signal. The sample is then excited with light at a wavelength absorbed by the donor fluorophore and the fluorescent emission from the fluorescence energy transfer pair is detected for the determination of that target. See U.S. Pat. No. 6,174,670.
U.S. Pat. No. 5,866,336 describes sunrise primers, that employ a hairpin structure that is similar to molecular beacons with a fluorescent and quenching moiety, but have a target binding sequence which serves as a primer. During amplification, when the primer's complementary strand is synthesized, the hairpin structure is disrupted, and the quencher is removed from the fluorophore, generating a signal.
Scorpion probes combine a primer with a hairpin structure and contain a sequence that is complimentary to the target sequence. When in the hairpin structure, a quencher moiety is effective in quenching a fluorophore moiety. The hairpin structure is disrupted by the binding of the probe region to the target sequence amplicon attached to the primer, and the quencher moiety is physically removed from the fluorophore, and fluorescent signal can be detected. There are several advantages of such intramolecular reactions over intermolecular probes. Intramolecular hybridization is fast and is not a limiting step, even with the fastest PCR protocols. The probe element is stabilized by the intramolecular reaction, increasing probe melting temperatures by about 5-15° C., so that shorter probes can be used, and in the hairpin or stem-loop format, a single oligonucleotide serves both as one of the primers and as a probe.
These and other methods can be used to detect target sequences, but they all are designed for specific sequences and have expensive detection moieties attached. A method that overcomes some of these issues, particularly for genotyping, is Snapback single strand conformation polymorphism, or “SSCP”, which uses a primer of a specific sequence to introduce secondary structure into PCR products that are then separated by electrophoresis. For example, a complementary 8-11 bp primer tail loops back on its complementary sequence in the extension product, creating a hairpin in the single stranded amplicon, which is later detected by gel separation. This method relies on post-amplification gel separation, which is not a technique that can easily transfer to clinical settings, and the additional step increases the time and costs of the assay.
Sequencing is currently the gold standard for identifying sequence variation. Even though costs are decreasing, sequencing is still a complex process that is not rapid, simple, or inexpensive when applied to specific genetic diagnosis or pharmacogenetics. Standard sequencing requires seven steps: 1) amplification by PCR, 2) clean up of the PCR product, 3) addition of cycle sequencing reagents, 4) cycle sequencing for dideoxy termination, 5) clean up of the termination products, 6) separation by electrophoresis, and 7) data analysis. This complexity can be automated and has been in some sequencing centers, but sequencing still remains much more complex than the methods of the present invention. Further, when large or multiple genes are analyzed, often over 90% of the sequenced products come back normal.
Moreover, current sequencing methods fail to identify low copy alleles, particularly when the alleles are present in an allele fraction of less than 20%. Identifying the presence of these low-copy alleles is important in a number of settings, illustratively in identifying the presence of certain oncogene mutations or changes in tumor samples or peripheral fluids such as blood. The presence or absence of such alleles can be particularly important for the selection of treatment protocols, illustratively with detection/confirmation of common somatic mutations (p53, EGFR, BRAF) and early identification of mutant bacterial infections (e.g., malaria) where standard therapies are contraindicated. Other examples of low levels of alleles that can be found against a predominant background are in mitochondrial DNA and fetal DNA present within maternal circulation. In addition, detection of low levels of epigenetic mutations is desired. For example, it was recently found that BRCA1 promoter methylation between 1 and 10% was associated with certain breast cancer phenotypes (Snell et. al., 2008, Breast Cancer Research)
PCR-based techniques for enriching the proportion of minority alleles and mutations in a sample are known. When the genotype of the mutation is unknown, COLD-PCR can be used (Li J, et al., Nat Med 2008; 14:579-84). It is known that this technique can detect down to a 1:100 ratio of mutant allele to wild type. However, because COLD-PCR is nonspecific and detects any variant that occurs, additional analysis is necessary to identify the products. For enriching known SNPs, some of the most popular techniques are ARMS (Newton C R, et al., Nucleic Acids Res 1989; 17:2503-16), PNA-mediated PCR (Nielsen PE, et al., Science 1991; 254:1497-500; Dabritz J, et al., Br J Cancer 2005; 92:405-12), LNA-mediated WTB-PCR (Dominguez P L, Kolodney M S. Wild-type blocking polymerase chain reaction for detection of single nucleotide minority mutations from clinical specimens. Oncogene 2005; 24:6830-4), MAMA-PCR (Cha R S, et al., PCR Methods Appl 1992; 2:14-20), TaqMAMA (Li B, et al., Genomics 2004; 83:311-20; Easterday W R, et al., Biotechniques 2005; 38:731-5), and SCORPION® primers (Whitcombe D, et al., Nat Biotechnol 1999; 17:804-7). These methods detect mutations by allele specific PCR, noting differences in quantification cycle (ΔCq) and can detect a 1:1000 ratio of mutant allele to wild type.
In ARMS PCR (or PCR amplification of specific alleles (PASA)), one of the primers is designed in such a way that it is able to discriminate among templates that differ by a single nucleotide residue located at the 3′-end of that primer. Only that sequence that matches the 3′-end of the primer is extended efficiently. Thus, an ARMS primer can be designed to amplify a specific member of a multi-allelic system while remaining refractory to amplification of another allele that may offer by as little as a single base from the non-complementary allele.
High resolution melting was introduced as a homogeneous method of scanning PCR amplicons for heterozygous sequence variants. See, e.g., U.S. Pat. Nos. 7,387,887 and 7,582,429, herein incorporated by reference in their entirety. Based on the use of dsDNA saturating dyes, high resolution melting is capable of detecting SNPs and insertions/deletions in amplicons up to 400 bp at a sensitivity >99%. Since its introduction in 2003, additional applications for high resolution melting have been developed, including genotyping for known sequence variants using small amplicons or unlabeled probes (LUNAPROBES™) Unlabeled probes are blocked on the 3′-end to prevent extension during PCR and may use a dsDNA saturation dye, illustratively LCGREEN® Plus (Idaho Technology, Salt Lake City, Utah), to discriminate the genotype of the allele based on probe melting temperature (Tm). The probe sequence can be designed to match either allele and is based on maximizing the ΔTm between the perfect match and mismatched probe. For more information on the use of unlabeled probes, see U.S. Pat. No. 7,387,887, already incorporated by reference.
Snapback primers may also be used for genotyping with or without high resolution melting. With a Snapback primer, the primer comprises a probe element specific for a locus of the target nucleic acid and a template-specific primer region, wherein the probe element is 5′ of the template-specific primer region. After amplification, the probe element may hybridize to the locus to form a hairpin in an intramolecular reaction or may hybridize to its complement strand in an intermolecular reaction. Thus, a Snapback primer incorporates the probe element into the same oligonucleotide as the primer. Snapback primers may be labeled, but they are often used unlabeled, in a manner similar to unlabeled probes. See WO 2008/109823, incorporated herein in its entirety, for a detailed discussion of Snapback primers.
It has been found that the probes themselves may be used to bias amplification of low fraction alleles. With minor allele amplification bias (MAAB) techniques, the probe (whether unlabeled probe, Snapback probe element, or other probe) is matched to the higher fraction allele, and “allele amplification bias” is empirically determined by setting the annealing temperature (or extension temperature, if used) of PCR somewhere between the Tm of the perfectly matched and somewhat below the Tm of the mismatched probe. At this mid-Tm annealing temperature, the perfectly matched probe is bound to its target (often the wild type allele) and is stable enough to retard amplification, while the probe melts off of the mismatched allele and extension may proceed unhindered. An exo− polymerase may also be used to avoid probe digestion and aid in biasing amplification of the lower Tm allele. See WO 2010/054254, incorporated herein in its entirety.